Inflammatory diarrheal illnesses are a major public health concern and economic burden in the United States and worldwide. Adenosine is an endogenous signaling molecule whose level is increased in the colonic tissue and luminal fluid during active inflammation. In the intestine, the adenosine 2B receptor (A2BAR) is the predominant adenosine receptor expressed in colonic epithelial cells and mediates the effects of adenosine. Our research, supported by NIH K08 and R01 funding, has focused on characterizing the signaling mechanisms and biological function of colonic epithelial A2BAR. This competing renewal application is built on a series of in vitro as well as in vivo observations that demonstrate a pro-diarrheogenic and pro-inflammatory role for colonic epithelial A2BAR. Together, our studies demonstrate that the activation of A2BAR orchestrates acute inflammatory response by coordinating interactive signaling between epithelial cells and neutrophils: i) A2BAR is intracellular at rest and is recruited to the membrane upon agonist stimulation; ii) A2BAR mRNA and protein levels are upregulated during acute flares in human and animal models of inflammatory bowel disease (IBD); iii) A2BAR mediates apically polarized IL-6 and fibronectin secretion, which in turn mediate neutrophil activation and bacterial adhesion/invasion respectively; iv) A2BAR augments IL-8 secretion and mediates neutrophil recruitment in response to inflammatory stimuli; v) A2BAR antagonism or gene deletion inhibits neutrophil infiltration; vi) genetic ablation of A2BAR in mice protects against bacterial and chemically-induced diarrhea and inflammation; and vii) inhibition of A2BAR using highly specific inhibitors ameliorates acute and chronic inflammation in mice. Although we have made significant advances in understanding the role of epithelial A2BAR in orchestrating mucosal inflammatory response, the physiological role of A2BAR, its regulation during inflammation and the contribution of immune cell A2BAR in inflammation is unknown. The goal of the studies proposed herein is to elucidate the physiological function of the A2BAR, regulation of the colonic epithelial A2BAR expression, and determine the role of immune cell A2BAR in inflammatory diarrheal illnesses. We hypothesize that the A2BAR plays a central role in mediating fluid secretion in the normal colon. Upregulation of A2BAR expression and function in epithelial and immune cells during intestinal inflammation plays an important role in inflammatory response; therefore A2BAR antagonism is an effective treatment for inflammatory diarrheal illnesses. These proposed studies will not only resolve the questions concerning the physiologic significance of A2BAR in the regulation of colonic fluid secretion and inflammatory response, but will also provide proof-of-principle for the development of A2BAR-targeted therapies in an arena that offers limited treatment modalities for intestinal inflammatory disorders. PUBLIC HEALTH RELEVANCE: Inflammatory diarrheal diseases are a cause of significant morbidity and mortality in U.S. and worldwide. The pathogenesis of these illnesses is unknown. Adenosine, is a naturally occurring metabolite that is generated during active intestinal inflammation, and its levels increase during inflammation. In the intestine, adenosine 2b receptor (A2B receptor) mediates the effects of adenosine. This proposal will elucidate the function, regulation and biological actions of adenosine 2b receptor in diarrhea and inflammation. Importantly, information gained from this project will be instrumental for the generation of new strategies aimed at preventing and/or treating intestinal inflammatory and diarrheal illnesses.